What is happening to my 18S? - (Jun/08/2007 )

Hi everyone,

I noticed something strange with my 18S internal standard sample in my RT-QPCR runs. The intensity drops off after a while while all other samples climb and plateau. A friend suggested that it was possibly condensation on the sides of the tubes or the caps popping off the tube during the run that can cause this. However, I have not noticed either of that happening. I've attached a graph below. Any insight would be much appreciated!

1ug mRNA is RT in 20uL reaction2uL of RT reaction is used in a template for a 20uL Real Time PCR reaction (one for each target gene)

-TonyL-

How the raw data look like? (e.g. without baseline substraction and other things)

-Trof-

Hi Trof,

This is the graph without baseline subtraction:

So does that mean my PCR baseline is increasing during the run for 18S but not for other samples?

-TonyL-

I don't think the baseline is actually increasing. Clearly your problem is very high concentration of 18S, so the log-linear phase starts early. There is actually no baseline..

Programs determine baseline in some of the early cycles (for example 2-6 cycle) if there is some increase in that interval, it's substracted as a baseline and distorts the graph as on your first image. Sometimes it sould help setting baseline to earlier cycles, if possible, but generally visible increase before 4th cycle is bad.

You should dilute your samples or use a different housekeeping, 18S is a very abundant one.

-Trof-

Thank you for shedding light on that Trof! I've tried GAPDH instead and that works well so far.